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Not applicable.
The invention relates to assays in which intracellular chelators are used to alter the kinetics of signal generation triggered by divalent cations, particularly calcium.
A variety of cell-based functional assays utilize measurements of intracellular calcium concentration to evaluate the activity of proteins in a normal physiological environment. These include assay for G-protein coupled receptors (GPCR) and plasma membrane ion channels. The large and rapid increase in intracellular calcium concentration by stimulation of these receptors and ion channels can be detected by intracellular calcium-sensitive probes, including fluorescent dyes, calcium-binding proteins such as the bioluminescent protein, aequorin, and modified green fluorescent protein-calmodulin chimer (Ungrin, M. D., Singh, L. M. R., Stocco, R., Sas, D. E., Abramovitz, M. (1999) An automated aequorin luminescence-based functional calcium assay for G-protein-coupled receptors. Anal. Biochem. 272, 34-42; Takahashi, A., Camacho, P., Lechleiter, J. D., and Herman, B. (1999) Measurement of intracellular calcium. Physiological Reviews 79, 1089-1125; Gonzalez, J. E., Oades, K., Leychis, Y. Harootunian, A., and Negulescu, P. (1999) Cell-based assays and instrumentation for screening ion-channel targets. Drug Discovery Today 4, 431-439; Miyawaki, A., Liopsi, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M., and Tsien, R. Y. (1997) Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 388, 882-887; Creton, R., Kreiling, J. A., and Jaffe, L. F. (1999) Calcium imaging with chemiluminescence. Microsc. Res. Tech. 46, 390-397; Prasher, D., McCann, R. O., and Cormier, M. J. (1985) Cloning and expression of the cDNA coding for aequorin, A bioluminescent calcium-binding protein. Biochem. Biophys. Res. Commun. 126, 1259-1268).
The detection of rapid and transient changes in intracellular calcium concentration requires instrumentation that simultaneously mixes a stimulant with cells and immediately detects the change in fluorescence or luminescence from the calcium reporter. This requirement for simultaneous liquid dispensing and signal detection has limited the application of these calcium transient measurements in cell-based functional assays due to lack of affordable instrumentation. The present invention demonstrates the utility of an intracellular calcium chelating agent to delay and/or prolong the calcium signaling response in a high density screening format such as 96-well, 384-well, 1526-well or 3456-well microtiter plates. The altering of the kinetics of the calcium signal in the presence of the chelator maintains the integrity of the assay while providing greater time for measuring the transient signal after addition of the stimulant. Therefore high throughput screening functional assays can also be performed on a greater variety of laboratory instruments.
Practitioners in the art of screening have been attempting to provide for methods to extend the time available to measure fluorescent or luminescent signals. For example, one idea was to add a reagent to alter or slow the kinetics of a cellular reporter enzyme reaction. Examples of this approach are the transformation of flash luciferase-based luminescence to glow-luminescence formats such as the PACKARD LUCLITE(copyright) Luciferase Reporter Gene Assay Kit (see U.S. Pat. No. 5,618,682 and EPO Patent Application 94 102 080.2) or PROMEGA STEADY-GLO(trademark) Luciferase Assay System (see U.S. Pat. No. 5,283,179).
Intracellular chelators of calcium are used routinely to eliminate calcium responses in a number of systems but the kinetics of intracellular calcium responses have primarily been described in the absence of chelators (Takahashi, A., Camacho, P., Lechleiter, J. D., and Herman, B. (1999) Measurement of intracellular calcium. Physiological Reviews 79, 1089-1125; Struk, A., Szucs, G., Kremmer, H., and Melzer, W. (1998) Fura-2 calcium signals in skeletal muscle fibres loaded with high concentrations of EGTA. Cell Calcium 23, 23-32; Keller, J. N., Guo, Q., Holtsberg, F. W., Bruce-Keller, A. J., and Mattson, M. P. (1998) Increased sensitivity to mitochondrial toxin-induced apoptosis in neural cells expressing mutant presenilin-1 is linked to perturbed calcium homeostasis and enhanced oxyradical production. J. Neurosci. 18, 4439-4450). However, in attempts to understand the neuro-protective properties of BAPTA-AM, this chelator""s effects on intracellular calcium transients has been studied in some detail (Tymianski, M., Wallace, M. C., Spigelman, I., Uno, M., Carlen, PI L., Tator, Ch H., and Charlton, M. P. (1993) Cell-permeant Ca2+ chelators reduce early excitotoxic and ischemic neuronal injury in vitro and in vivo. Neuron 11, 221-235). For example, when high concentrations of glutamate were used to stimulate mouse spinal cord neurons, BAPTA-AM was protective, and primarily affected the fast transient by decreasing the amplitude and delaying the rise and decay time of the transient (Tymianski, M., Charlton, M. P., Carlen, P. L., and Tator, C. H. (1994) Properties of neuroprotective cell-permeant Ca2+ chelators: Effects on [Ca2+]i and glutamate neurotoxicity in vitro. J. Neurophys. 72, 1973-1992). Similar results were obtained using ionomycin-induced calcium influx where observed calcium transients were up to 8-fold slower in the presence of BAPTA-AM (Collatz, M. B., Rudel, R., and Brinkmeier H. (1997) Intracellular calcium chelator BAPTA protects cells against toxic calcium overload but also alters physiological calcium responses. Cell Calcium 21, 453-459).
It has now been discovered that buffering intracellular calcium with a calcium binding reagent can delay and/or prolong the calcium signaling response in a manner that can be practically applied to high throughput screening assays.
The present invention provides assays in which intracellular chelators are used to alter the kinetics of signal generation triggered by divalent cations, particularly calcium accumulation in the cell cytoplasm. The use of an intracellular chelator in the assay can delay and prolong the signal, allowing the signal to be detected by automated instrumentation without the need for simultaneous liquid handling at the time of detection.
An aspect of this invention is a high throughput assay in which cells having a receptor, which upon activation induces a calcium influx into the cell, are contacted with an intracellular calcium chelator. In preferred embodiments the chelator is 1,2-bis(o-aminophenoxy)ethane-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA-AM). In particular embodiments, the cells also contain an aequorin-based or fluorescent dye-based or green fluorescent protein-calmodulin chimer- based signal generation system. In a preferred embodiment, the assay is conducted in a microtiter plate format and an instrument is used to detect the signal in each well.
By xe2x80x9caboutxe2x80x9d it is meant within 10% to 20% greater or lesser than particularly stated.
As used herein an xe2x80x9cagonistxe2x80x9d is a compound or molecule that interacts with and stimulates an activity of a receptor.
As used herein an xe2x80x9cantagonistxe2x80x9d is a compound that interacts with a receptor and inhibits or interferes with the activation of the receptor.
As used herein an xe2x80x9cinhibitorxe2x80x9d is a compound that interacts with and inhibits or prevents the activation of a receptor.
All of the references cited herein are incorporated by reference in their entirety as background material.